DESCRIPTION: Although it is well established that receptor-mediated elevation in diacylglycerol (DAG) levels leads to activation of protein kinase C (PKC), a novel receptor for this lipophilic second messenger and its analogs, the phorbol ester tumor promoters, was recently discovered: chimaerin. In contrast to PKC, N-chimaerin does not possess a kinase domain. Its catalytic domain has high homology to BCR (breakpoint cluster region), a protein involved in Philadelphia chromosome translocation in chronic myelogenous leukemia. Like BCR, n-chimaerin has GTPase-activating protein (GAP) activity for the p21Rac, a small GTP-binding protein which plays a central role in cytoskeletal structure, gene transcription, cell growth and malignant transformation. The expansion of the chimaerin family with the cloning of new isoforms (a1- or "n-",a2-,b1-,andb2-chimaerins) indicates that a high degree of complexity may exist in the downstream pathways triggered by DAG. The fact that the a2- and b2-chimaerins possess SH2 motifs on their structure predicts that these chimaerin isoforms may associate with tyrosine phosphorylated proteins which may regulate their activity or localization, therefore suggesting crosstalk between different signaling pathways. The overall goal of this proposal is to evaluate whether the second messenger DAG and the phorbol ester tumor promoters regulate the chimaerin activity in addition to that of PKC, and to determine the biological consequences of the activation of this pathway in the cell. In Specific Aim 2, he will identify proteins that associate to chimaerins. The SH2 domains of a2- and 2-chimaeri withphosphotyrosine proteins. We will also study whether translocation induced by phorbol esters leads to the association of chimaerins with specific targets or anchoring proteins. In Specific Aim 3 he will focus on the biological roles of chimaerins. Our hypothesis is that chimaerins may affect those responses mediated by p21Rac, and therefore affect cell growth, malignant transformation, and cell morphology.Dr. Kazanietz's research has the potential for defining new "PKC independent" pathways for the phorbol ester tumor promoters and DAG, and hopefully will yield new insights into the events controlling malignant transformation. The characterization of the targets for the phorbol esters would be valuable to clarify their biological actions and also to unravel pathways involved in the mechanisms of carcinogenesis.